The intervertebral disc (IVD) separates the vertebrae of the spine and functions to resist loading that the spine is subjected to during daily life. It has a unique structure with an inner water-rich gel-like nucleus pulposus (NP) with random organization of extracellular matrix (ECM); an outer fibrous annulus fibrosis (AF) with well-organized collagen sheets; and thin cartilaginous end-plates supplying nutrients to the disc. Normal disc function is enabled by the special configuration and differential hydration properties of NP and AF. The NP is predominately in proteoglycans, which is hydrophilic, and therefore maintains a more than 80% hydration, providing high hydrostatic pressure to resist loading. The AF also contains more than 60% water and is predominately in closely-packed collagen providing strong tensile strength and assisting the NP in resisting loading. Disc cells are chondrocyte-like cells able to produce ECM in particular proteoglycans so as to maintain the hydration properties of IVD.
Disc degeneration is a common clinical problem affecting human populations worldwide, causing low back pain and limited mobility. Although the pathogenesis is not completely known, structural and compositional changes of degenerated discs are extensively characterized. In early disc degeneration, disc cells, in particular the NP cells, become less capable of synthesizing ECM, the gelatinous NP becomes more fibrous and therefore reduces the water content of the disc. As the disease progresses, there is more ECM and structural changes such as proteoglycan and collagen degradation, reducing disc height. At advanced stages, structural collapse and eventual loss of disc function in resisting loading result.
There is no satisfactory clinical treatment for advanced disc degeneration. Spinal fusion does not solve the problem but does relieve pain. However, the fused spine loses motility and is non-functional. The only clinically available option for replacement are artificial discs made of metal and rubber whose purpose is to preserve the motion between the vertebrae. However, these prostheses cannot integrate with the surrounding tissues and allow new tissue formation. Prosthetic failure and high re-operation rate has been reported (Enker, et al. Spine, 18(8):1061-70 (1993), Griffith, et al., Spine, 19(16):1842-9 (1994)).
Research and development efforts to restore disc function have been extensive, ranging from administration of growth factors, for example, multiple injections of transforming growth factor-beta (TGF-b), insulin-like growth factor-1 (IGF-1), and basic fibroblast growth factor (bFGF), to stimulate disc cell secretion of ECM (Walsh, et al., Spine, 29(2):156-63 (2004)); gene therapy to deliver cDNA coding for several growth factors stimulating ECM synthesis (Wallach, et al. Spine, 28(15 Suppl):S93-8 (2003)) and cell therapy where mature autologous disc cells, chondrocytes, or stem cells are transplanted to the intervertebral disc to replenish the cells and enhance ECM production (Brisby, et al., Orthop. Clin. North Am., 35(1):85-93 (2004)). These methods are for early stage degeneration where the disc is still functional and retains its structural integrity, not in advanced degenerative cases, where structural and functional replacement is needed.
One approach to treating advanced disc degeneration is to replace a non-functional disc with a tissue engineered substitute resuming the disc function immediately after implantation, integrating with the surrounding tissues and maintaining its function. In general, the substitute consists of a scaffold, which provides structural and functional support with good stability for a substantial amount of time to allow new tissue growth; cellular components embedded in the scaffolds, they are originated from the patient's own cell sources that are able to take up normal disc cells' job in synthesizing and regulating new ECM in response to the local milieu so as to maintain the disc structure and function; and growth-stimulating signals, corroborating with the local milieu, which could be biological and physical, to guide the cellular components to perform appropriately.
Seeding cells onto or into pre-cast scaffolds is the approach that dominates the field of tissue engineering. Porous hybrid materials such as bioactive glass and synthetic polymers such as D,L poly(lactide-co-glycolide) (PLGA) have been used as the substrates and surgically inserted into degenerated discs together with cells extracted from the nucleus, as discussed in U.S. Pat. Nos. 5,964,807 and 6,240,926. U.S. Pat. No. 6,723,335 discloses using decellularized IVD nucleus fluid from donor vertebrate for seeding of living cells from the donor, after stabilization using photooxidizing crosslinking. Atelocollagen scaffold has been developed to replace the NP (Sato et al., Med. Biol. Eng. Comput., 41(3):365-71 (2003), Sato, et al., J. Biomed. Mater. Res. A. 64(2):248-56 (2003)). Allograft disc cells demonstrated good scaffold biocompatibility in supporting proliferation and ECM production. The biodegradable synthetic polymer poly-glycolic-acid (PGA) was used to replace the AF and alginate hydrogel to replace the NP after loading with disc cells (Mizuno, et al., Spine, 29(12):1290-7 (2004)).
The distribution of cells depends on the penetration and migration of the cells into the scaffolds. Unfortunately, penetration of the cells into preformed scaffolds is usually limited to the surface (Seguin, et al., Spine, 29(12): 1299-306 (2004)). The penetration of cells into scaffolds also depends on the pore size of the scaffolds. Large size ensures better penetration but compromises the mechanical properties. Efforts such as agitation during seeding and creating channels in the scaffolds have improved the amount of cells reaching the half thickness of the scaffolds to around 38% (Rose, et al., Biomaterials, 25(24):5507-14 (2004)) but the high speed agitation detrimentally affects cell viability and wide channels detrimentally affect the scaffold properties. Another limitation of the cell-seeding approach is that the distribution of cells in the scaffolds is not homogenous, which may affect the quality of the engineered tissue structures.
Mechanical properties of these scaffolds are not described but the ECM synthesized was far less than required to provide sufficient mechanical support as in the native discs (Masuda, et al., Spine, 29(23):2757-69 (2004)). Taking the mechanical requirement into consideration, there is an attempt to use bone substitute, calcium polyphosphate powder to develop scaffold for NP replacement (Seguin, et al., Spine, 29(12):1299-306 (2004)). The scaffold allows attachment of disc cells with enhanced proliferation and ECM production but the attachment was only limited to its surface. Further, the compressive modulus of the structures was far less than 100 KPa, only a minor portion of that in the native discs (Urban, et al., Spine, 29(23):2700-9 (2004)), reported to range from 3 to 31 MPa in humans (Elliott & Sarver. Spine, 29(7):713-22 (2004)). As a result, the above mentioned approaches may not have sufficient amount of extracellular matrix deposition to provide the necessary mechanical support for intervertebral disc.
Finally, the cell-seeding approach using preformed scaffolds can not be used for production of tissue with heterogeneous structures, consisting of different cell type and intensity, and with different ECM type and intensity, such as invertebral discs. U.S. Pat. No. 6,783,546 discloses a heterogeous structure with a keratin hydrogel sandwiched between layers of synthetic polymers such as silicone and polyethylene for breast reconstruction and NP replacement. However, these synthetic materials have limitations in biocompatibilities. No mechanical properties were reported. The processing of the keratin hydrogel includes heating to temperatures, such as 90° C., which is above the protein denaturation and cell damage so that no living components can be included during the fabrication process. As a result, better fabrication approach for IVD tissue engineering addressing the limitations of the cell-seeding-on-preformed-scaffold approach is warranted.